Mechanisms restraining the accumulation of antibody secreting cells
Lead Research Organisation:
Babraham Institute
Department Name: Immunology
Abstract
The B lymphocyte is the only immune cell type capable of development into the antibody secreting cell (ASC) a cell type commonly referred to as the plasma cell. Defining the genes and cellular processes that regulate ASC development, lifespan and what regulates the amount of antibody secreted by an ASC are fundamental questions -with answers that are likely to be relevant to many other dynamic cell systems in the body. The practical application of this knowledge is of direct relevance to the development of vaccines, interventions for autoimmune/allergic conditions, the impact on healthy ageing of abnormally increased numbers of ASC and the production of synthetic biological therapeutics by mammalian cells.
B lymphocyte activation and the pathway of ASC development is underpinned by dynamic changes in gene transcription coupled to post-transcriptional regulation of mRNA localisation, translation and stability. These act together to determine, where, when and in what amounts proteins are produced. The mechanisms of translational control of ASC function and survival is an area ripe for further discoveries in the emerging and important areas of frontier bioscience: these include linking the molecular biology of stress responses to cell differentiation; the avoidance of proteotoxicity; and the determinants of cellular longevity. Knowledge of these may have relevance to almost all biological systems and practical application in biotechnology or medical contexts.
This project builds on a new genetic screen for RNA binding proteins (RBP) that regulate the development and survival of ASC. The screen identified genes that were required for the accumulation of ASC and also genes which, when deleted, led to increased ASC formation and survival. The findings of this screen have been published on a preprint server, and this grant proposal is seeking to understand the in vivo immunology, the cell biology and molecular mechanism of modules within two multiprotein complexes [CCR4NOT and EIF3] known to be fundamental for transcription, RNA decay and translation.
Do these modules limit ASC development or survival, or do they affect both in vivo? Are the CCR4NOT and EIF3 modules coupled physically and functionally to each other in ASC to form a continuum that connects transcription mRNA stability and translation? Are they regulating homeostatic stress responses such as ER and oxidative stress that is fundamental for the secretory output of ASC? Do they mediate a mechanism to interlink proteostasis with nutrient sensing and oxidative sensing pathways? The module components are encoded by genes only found in higher eukaryotes and thus likely to be important for the unique biology of multicellularity.
We propose to study these genes using methods that evaluate quantitatively ASC formation and function in vivo as this is the powerful physiological system of choice. We propose to combine this with in vitro studies of cell biology informed by integrative data-driven transcriptomics methods that take into account the abundance and turnover of mRNA and its translation efficiency. Furthermore, we will determine the composition and location of the multiprotein complexes in which the protein encoded by the genes that limit ASC function reside. This combination of approaches can provide mechanistic insight into how the different processes which control gene expression are coupled to regulate cell function and homeostasis.
B lymphocyte activation and the pathway of ASC development is underpinned by dynamic changes in gene transcription coupled to post-transcriptional regulation of mRNA localisation, translation and stability. These act together to determine, where, when and in what amounts proteins are produced. The mechanisms of translational control of ASC function and survival is an area ripe for further discoveries in the emerging and important areas of frontier bioscience: these include linking the molecular biology of stress responses to cell differentiation; the avoidance of proteotoxicity; and the determinants of cellular longevity. Knowledge of these may have relevance to almost all biological systems and practical application in biotechnology or medical contexts.
This project builds on a new genetic screen for RNA binding proteins (RBP) that regulate the development and survival of ASC. The screen identified genes that were required for the accumulation of ASC and also genes which, when deleted, led to increased ASC formation and survival. The findings of this screen have been published on a preprint server, and this grant proposal is seeking to understand the in vivo immunology, the cell biology and molecular mechanism of modules within two multiprotein complexes [CCR4NOT and EIF3] known to be fundamental for transcription, RNA decay and translation.
Do these modules limit ASC development or survival, or do they affect both in vivo? Are the CCR4NOT and EIF3 modules coupled physically and functionally to each other in ASC to form a continuum that connects transcription mRNA stability and translation? Are they regulating homeostatic stress responses such as ER and oxidative stress that is fundamental for the secretory output of ASC? Do they mediate a mechanism to interlink proteostasis with nutrient sensing and oxidative sensing pathways? The module components are encoded by genes only found in higher eukaryotes and thus likely to be important for the unique biology of multicellularity.
We propose to study these genes using methods that evaluate quantitatively ASC formation and function in vivo as this is the powerful physiological system of choice. We propose to combine this with in vitro studies of cell biology informed by integrative data-driven transcriptomics methods that take into account the abundance and turnover of mRNA and its translation efficiency. Furthermore, we will determine the composition and location of the multiprotein complexes in which the protein encoded by the genes that limit ASC function reside. This combination of approaches can provide mechanistic insight into how the different processes which control gene expression are coupled to regulate cell function and homeostasis.
Technical Summary
To resolve the consequences of the mutation of CNOT and EIF3 module components for development and survival in vivo we will electroporate Cas9 ribonucleoproteins into naïve antigen-specific B cells and transfer them into mice which will be challenged with specific antigen. By performing in vitro studies on the same batches of cells we can establish a deep understanding of the effects of the mutations on the cell biology of ASC.
To establish molecular mechanisms, we will apply SLAM-seq, ribosome profiling, proteomics in a comparison between control and mutant cells. This will allow us to get at the key question of how the module components connect transcription, RNA decay and translation. We will combine this data with data that we will generate using the recently developed "improved-individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation" (iiCLIP) method and data driven approaches that honed in my lab. This approach will give insight into molecular mechanism and inform the refinement of the studies on the cell and in vivo biology.
The physical connection between CNOT and EIF3 modules and the existence of spatially discrete complexes will be tested using BIO-ID proximity labelling. In addition to testing our hypothesis that the modules can associate, this experiment can identify unanticipated protein partners that bring RNAs to the multiprotein complexes or mediate additional effector functions of the complex.
Our integrative approaches will provide a deep mechanistic insight into the regulation of ASC biology and gene expression by the CNOT and EIF3 modules.
To establish molecular mechanisms, we will apply SLAM-seq, ribosome profiling, proteomics in a comparison between control and mutant cells. This will allow us to get at the key question of how the module components connect transcription, RNA decay and translation. We will combine this data with data that we will generate using the recently developed "improved-individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation" (iiCLIP) method and data driven approaches that honed in my lab. This approach will give insight into molecular mechanism and inform the refinement of the studies on the cell and in vivo biology.
The physical connection between CNOT and EIF3 modules and the existence of spatially discrete complexes will be tested using BIO-ID proximity labelling. In addition to testing our hypothesis that the modules can associate, this experiment can identify unanticipated protein partners that bring RNAs to the multiprotein complexes or mediate additional effector functions of the complex.
Our integrative approaches will provide a deep mechanistic insight into the regulation of ASC biology and gene expression by the CNOT and EIF3 modules.
